The single-electron reduction of molecular oxygen by members of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family of complex enzymes is an initial step in the generation of reactive oxygen species (ROS) important for a number of biochemical and biological functions. The best studied of the NADPH oxidases, namely that of the peripheral blood phagocytes plays an essential role in the host defense against microbial pathogens. Formation of the stimulus-activated phagocyte NADPH oxidase enzyme complex of NOX species NOX1-4 involves the association of at least four cytosolic subunits: p47phox, p67phox, p40phox, and the small GTPase Rac2; with a core membrane-bound catalytic subunit, flavocytochrome b558that includes a heterodimer of NOX2 (also known as gp91phox) and p22phox. With the exception of p40phox, there is an absolute requirement for each of the subunits for the formation of a functional NOX2 enzyme in vivo. Critical intermediate steps in the activation process are the phosphorylation of multiple serine residues in p47phox, which reverses its auto-inhibited state, revealing binding sites for flavocytochrome b558, and the activation of Rac2 by the exchange of GDP with GTP. The full assembly of an active phagocyte NADPH oxidase complex is dependent on a number of specific protein binding domains located on each of the subunits (1-3).
The recent discovery in non-phagocytic cells of oxidase core proteins homologous to NOX2 has expanded knowledge of both the role of ROS in biochemical and cellular processes, and the mechanisms of activation of the NOX core proteins (4-11). All NOX family members share the common features of multiple membrane-spanning domains and binding sites for heme, FAD, and NADPH. However it is now emerging that the NOX proteins differ in their modes of activation and requirements for cytosolic cofactors. NOX5, expressed in spermatocytes and lymphoid tissues, has an extended N-terminal domain containing four Ca2+-binding, EF-hand motifs (8), Binding of Ca2+ by these sites leads to a conformational change in the protein sufficient for the formation of a functional oxidase, apparently without cofactor requirements (12). NOX4 displays a rather wide distribution in tissue, but is particularly well expressed in renal cells (6-10; 13) where it serves as an oxygen sensor regulating erythropoietin production. It has been postulated to serve as an oxygen sensor (6), and has been implicated in the signal transduction pathways mediating the angiotensin II response in glomerular mesangial cells (13), the insulin-receptor mediated response in adipocytes (14), and the endoplasmic reticulum stress response in human aortic smooth muscle cells (15). NOX3 is expressed in fetal kidney (9), and is particularly highly expressed in the inner ear of the mammals (11). NOX3-deficiency in mice is associated with the otoconia-deficient head-tilt phenotype (16). Unique among the NOX proteins, NOX3 appears to function well with either p47phox and p67phox or their recently described homologues, NOXO1 and NOXA1 (11; 17-20). On the other hand, the activation of NOX1,a NOX isoform most highly expressed in colon epithelium, requires NOXA1 and NOXO1, and shows only minimal activity with the cytosolic proteins p47phox and p67phox. It has been suggested that the NOX1/NOXO1/NOXA1 oxidase system functions in an antimicrobial capacity, as well as in the regulation of cell growth.
The discovery of the phox protein homologues NOXA1 and NOXO1 has also changed the perspective on how the NOX proteins may be functionally regulated (18-20). Similar to p47phox, NOXO1 contains an N-terminal inositide-binding PX domain and the characteristic tandem SH3 domains in its central region. However, the basic region in the C-terminal end responsible for intramolecular binding and the resting auto-inhibited state in p47phox is absent from NOXO1. This difference is likely the basis for the observation that the NOX1/NOXO1/NOXA1 system displays constitutive activity in some cell types, whereas it is inducible in others (18-20). NOXA1, like its homologue p67phox, contains in its N-terminal region the complex TPR domain, which is responsible for binding activated (GTP-bound) Rac, an activation domain containing a critical valine, a putative C-terminal PB1 domain and a C-terminal SH3 domain.
In addition to NOXA1, cytosolic cofactor p67phox (19) is also known to be active as a homologue to NOXA1 in the formation of NOX oxidases NOX1-4.
In spite of the considerable knowledge developed regarding NADPH oxidase enzymes the cell type distribution of NOX1-5 in various tissues, and a mechanism of cytochrome b558 activation, surprisingly little progress has been made in devising therapeutics that draw on this information. Thus, there exists a need for a NOX cytosolic cofactor having a biological activity different from the wild-type cofactor.